Method for detecting cool feeling intensity in cooling agent monomer based on cell membrane method and application

Abstract

The invention belongs to the technical field of tobacco chemistry, and particularly relates to a method and application for detecting cool feeling intensity in a cooling agent monomer based on a cell membrane method.The method comprises the four steps of cell culture, fluorescent dye loading, FLIPR detection and data analysis, CHO-TRPM8 cell lines expressing a TRPM8 channel are utilized, the FLIPR technology is combined, and the cool feeling intensity in the cooling agent monomer is detected. By monitoring the change of the concentration of calcium ions in cells in real time, establishing a dosage delta F / F0-reaction curve of the concentration-maximum fluorescence intensity change value of the cooling agent and calculating the half effective concentration EC50 of the cooling agent, the cool feeling intensity in the cooling agent is evaluated, and compared with a traditional method, the method has the advantages of being high in sensitivity, good in repeatability, high in flux and the like, and has a good application prospect. The cool feeling intensity can be rapidly and accurately measured, and the problems of poor subjectivity, poor repeatability and the like of traditional artificial sensory evaluation are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco chemistry, and particularly relates to a method for detecting the cooling intensity in a cooling agent monomer based on a cell membrane method and an application thereof. Background Art

[0002] With the continuous development of the tobacco products market, consumers have increasingly higher requirements for the sensory experience of cigarettes, among which coolness, as one of the important sensory indicators, has received extensive attention. As the key ingredient that gives cigarettes a cool feeling, the blending technology of cool flavors is crucial. In the entire blending process, the evaluation method of coolness intensity is of utmost importance, which directly affects the sensory quality of cigarette products and their competitiveness in the market. A cigarette with just the right coolness intensity can enhance consumers' smoking pleasure while establishing a good brand image.

[0003] However, the current traditional evaluation method of coolness intensity mainly relies on manual sensory evaluation. The specific operation is that professionally trained evaluation team members quantify the coolness intensity through sensory evaluation according to the standardized scoring system. However, this method has many disadvantages. On the one hand, due to the polymorphism of the human TRPM8 receptor gene, there are significant differences in the perception of coolness among individuals. Specifically, some people are more sensitive to coolness due to their genetic characteristics, while others are relatively dull, which leads to different evaluation personnel's coolness scores for the same cigarette sample may be very different. On the other hand, the ambient temperature and humidity have a significant impact on the results of manual evaluation. In a high temperature and high humidity environment, the human body's perception of coolness will be relatively weakened; while in a low temperature and dry environment, the coolness may be amplified. Based on the above factors, the coolness intensity evaluation results obtained by manual evaluation are highly subjective and have poor repeatability, which is difficult to meet the high standards of the modern tobacco industry for precise blending and strict quality control. In the modern tobacco industry, precise blending requires precise control of the amount and matching ratio of cool flavors to ensure that the coolness intensity of each batch of products is consistent, but the limitations of traditional evaluation methods make this goal difficult to achieve.

[0004] In order to solve the above problems, the present invention is proposed. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for detecting the cooling intensity in cooling agent monomers based on the cell membrane method, aiming to establish an objective evaluation system for cooling intensity and provide technical support for the precise formulation of cooling flavors for tobacco. The method uses the Chinese hamster ovary cell (CHO-TRPM8) cell line expressing the TRPM8 channel and the FLIPR technology to establish the cooling agent concentration-maximum fluorescence intensity change value ΔF / F by real-time monitoring of changes in intracellular calcium ion concentration.0 - Response curve, calculate the half effective concentration EC of cooling agent 50 , thereby evaluating the cooling intensity of cooling agents. It is also possible to compare the EC 50 The value is used to perform relative quantitative analysis of the cooling intensity of the cooling agent. Compared with the traditional method, the present invention has the advantages of high sensitivity, good repeatability and high throughput, and can achieve rapid and accurate determination of the cooling intensity, overcoming the problems of subjectivity and poor repeatability of traditional manual sensory evaluation.

[0006] In order to achieve the above object, the specific scheme adopted by the present invention is:

[0007] The first aspect of the present invention provides a method for detecting the cooling intensity of a cooling agent monomer based on a cell membrane method, the method comprising the following steps:

[0008] Step (1), cell culture:

[0009] Chinese hamster ovary (CHO) cells stably expressing TRPM8 receptor were seeded into each well of a flat-bottom microtiter plate and proliferated to a predetermined confluency under culture conditions;

[0010] Step (2), fluorescent dye loading:

[0011] Remove the cell culture medium in the flat-bottom microtiter plate of step (1), and add a certain volume of HEPES-buffered saline (HBPS) buffer containing 30 μg / ml of calcium ion fluorescent dye (Fluor-8) and 10 μg / ml of trypan red for fluorescent staining;

[0012] Step (3), FLIPR detection:

[0013] A cooling agent and a control sample of a certain test concentration are added to the wells of the flat-bottom microtiter plate in step (2), and the change in fluorescence signal intensity is continuously recorded using FLIPR, and analyzed using FLIPR Screen Works 3.1 software;

[0014] Step (4), data analysis:

[0015] The fluorescence signal recorded in step (3) is background corrected and normalized, and the maximum fluorescence intensity change value ΔF / F is calculated. 0 As a quantitative indicator of the cooling intensity, i.e. the percentage of TRPM8 channel activation, the dose ΔF / F of the cooling agent concentration-maximum fluorescence intensity change value was established. 0 - response curve, and then calculate the half effective concentration EC of the cooling agent 50 , based on the half effective concentration EC of cooling agents 50 Conduct relative quantitative analysis of cooling intensity, and use the half effective concentration EC 50The lower it is, the stronger the cooling sensation is.

[0016] TRPM8 receptor is a key molecule for the human body to perceive coolness, and can specifically respond to cooling substances such as menthol. As an efficient and sensitive cell function detection technology, FLIPR technology can quickly and accurately reflect the impact of external stimuli on cells by mainly monitoring the changes in intracellular calcium ion concentration in real time.

[0017] Preferably, the inoculation concentration of Chinese hamster ovary cells in step (1) is 2.0×10 4 ~4.0×10 4 cells / well, the culture medium is DMEM, and the culture conditions are 37°C, 5% CO2 and 95% relative humidity for 18-24 hours until the cell confluence is above 90%.

[0018] The main operation process of FLIPR Screen Works 3.1 software is as follows:

[0019] (1) Fluorescence data acquisition;

[0020] (2) The raw fluorescence data were exported as time-fluorescence intensity curves (F(t));

[0021] (3) Baseline correction: The mean fluorescence value of the 10 seconds before stimulation is taken as F 0 ;

[0022] (4)ΔF / F 0 Calculation: ΔF / F 0 =(FF 0 ) / F 0 , where F is the fluorescence intensity after stimulation;

[0023] (5) Built-in formula ΔF / F 0 =(FF 0 ) / F 0 Automatic generation of dose-response curves and calculation of EC 50 value.

[0024] If the Fluo-3 probe is enhanced 60–100 times, the signal dynamic range is ΔF / F 0 The values ​​are within the expected dynamic range of the probe, indicating that the receptor is stably expressed and functional.

[0025] Preferably, the volume of HBPS buffer added for fluorescent staining in step (2) is 30 to 50 μL, and the staining loading time ranges from 10 to 40 min.

[0026] Preferably, in the step (2), the mass percentage of the calcium ion fluorescent dye 30mmol / L, trypan red 10mmol / L, and HEPES buffered saline is 0.5%; the HEPES buffered saline contains: 10g / L HEPES; 16g / L NaCl; 0.74g / L KCl; 0.27g / L Na2HPO4.2H2O; and 2.0g / L dextrose.

[0027] Preferably, the cooling agent in step (3) includes L-menthol, menthone, menthyl acetate, WS-3, WS-5, WS-23, and diluted anhydrous ethanol; the control sample is anhydrous ethanol.

[0028] Among them, flat-bottom microtiter plates were used for experiments, which could process multiple sets of concentration gradients at a single time, significantly improving the detection efficiency.

[0029] The amount of the cooling agent added to the flat-bottom microtiter plate is 4-10 μL / well, and the dilution concentration of the cooling agent is set to 0.1-100 μg / mL.

[0030] Preferably, the detection parameters of the FLIPR are: Fluo-3 calcium ion probe, excitation wavelength 470-495 nm, emission wavelength 515-575 nm.

[0031] Preferably, the normalization method in step (4) is a min-max normalization method;

[0032] Dose of maximum fluorescence intensity change ΔF / F 0 The calculation formula is: ΔF / F 0 =(FF 0 ) / F 0 (F 0 is the initial fluorescence intensity, F is the fluorescence intensity after treatment)

[0033] The cooling agent concentration-maximum fluorescence intensity change value dose ΔF / F 0 -The reaction curve formula is: ΔF / F 0 =K·C, where K is the proportional constant, the value range is K>0, and C is the concentration of the cooling agent.

[0034] The second aspect of the present invention provides an application of the method of the first aspect in the development of a cool functional tobacco product by EC 50 The value can be used to calibrate the amount of cooling agent added to achieve precise control of the product's cooling properties.

[0035] The present invention has the following beneficial effects:

[0036] 1. The present invention innovatively constructs a method for detecting the cooling intensity of cooling agents based on the cell membrane method, which effectively solves the problem of subjective interference in traditional sensory evaluation methods, overcomes human judgment bias, and has the advantages of high sensitivity, good repeatability and high throughput. This method can quickly and accurately measure the cooling intensity.

[0037] 2. The present invention adopts the CHO-TRPM8 cell line expressing the TRPM8 receptor, and combines it with the FLIPR technology to accurately detect the cooling intensity of the cooling agent by real-time monitoring of the changes in intracellular calcium ion concentration, thereby establishing an objective evaluation system for cooling intensity, providing technical support for the precise formulation of cooling flavors for tobacco.

[0038] 3. The method of the present invention includes four steps: cell culture, fluorescent dye loading, FLIPR detection and data analysis. The operation is simple and scientific, and the experimental results are stable and reproducible. By calculating the half effective concentration EC of the cooling agent 50 , which provides a scientific basis for the amount and proportion of cooling agents added, and supports the precise control of the product's cooling properties.

[0039] 4. The method of the present invention can detect extremely low concentrations of cooling agents, with a detection limit of up to 0.03 g / mL, and has extremely high detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 = concentration of each cooling agent of the present invention - ΔF / F 0 Dose-response diagram;

[0041] Figure 2 The figure is a bar graph showing the sensory evaluation results of the cooling intensity of each cooling agent of the present invention. DETAILED DESCRIPTION

[0042] The present invention is further described below by way of examples, but is not limited to these examples.

[0043] The experimental methods without specific conditions in the examples are usually carried out under conventional conditions and conditions described in the manual, or under conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all commercially available unless otherwise specified.

[0044] like Figure 1 The figure shows the method of the present invention for the treatment of 6 cooling agents at different concentration gradients and ΔF / F 0 Dose-response curve relationship diagram, wherein Examples 1 to 4 list the testing methods of 6 cooling agents at 4 concentrations respectively.

[0045] Example 1

[0046] (1) Cell culture: CHO cells expressing the TRPM8 receptor clone were cultured at a concentration of 2.0 × 10 4 Cells / well were seeded in 384-well flat-bottom microtiter plates. CHO cells were cultured overnight at 37°C, 5% CO2 and 95% relative humidity.

[0047] (2) Fluorescent dye loading: The growth medium in the CHO-TRPM8 cell culture medium in (1) was removed and replaced with 30 μL of HEPES-buffered saline (HBPS) solution containing 5 μL Fluor-8 and 5 μL trypan red (10 mmol / L) at 37°C for 10 min, followed by fluorescent staining for 30 min.

[0048] (3) FLIPR detection: Add L-menthol (or menthone, menthyl acetate, WS-3, WS-5, WS-23, or anhydrous ethanol control sample) at a test concentration of 0.5 μg / mL to the wells of the flat-bottom microtiter plate containing HBPS buffer in step (2), record the fluorescence change value by FLIPR at an excitation / emission wavelength of 470 / 515 nm, and analyze it using FLIPRScreen Works 3.1 software. Wherein L-menthol (or menthone, menthyl acetate, WS-3, WS-5, WS-23, or anhydrous ethanol control sample) occupies all wells of the flat-bottom microtiter plate.

[0049] (4) Data analysis: The raw fluorescence signal recorded in (3) was subjected to background correction and min-max normalization, and then the maximum fluorescence intensity change value (ΔF / F 0 ) is used as a quantitative indicator of the intensity of the cooling sensation, namely the percentage of TRPM8 channel activation. By establishing a dose-response curve of the percentage of channel activation of the cooling agent concentration, the half effective concentration EC of the cooling agent can be calculated. 50 By comparing the EC of different cooling agents 50 The values ​​were used to conduct relative quantitative analysis of the cooling intensity.

[0050] The EC values ​​of this method for L-menthol, menthone, menthyl acetate, WS-3, WS-5, and WS-23 were calculated by changing the fluorescence intensity of different cooling agent solutions. 50 The values ​​were 8.8μg / mL, 9.3μg / mL, 8.2μg / mL, 9.1μg / mL, 8.0μg / mL, and 7.8μg / mL, respectively; the detection limits were 0.09μg / mL, 0.13μg / mL, 0.18μg / mL, 0.29μg / mL, 0.19μg / mL, and 0.09μg / mL, respectively.

[0051] Example 2

[0052] (1) Cell culture: CHO cells expressing the TRPM8 receptor clone were cultured at a concentration of 3.0 × 10 4 Cells / well were seeded in 384-well flat-bottom microtiter plates. CHO cells were cultured at 37°C and 5% CO 2 and 95% relative humidity overnight.

[0053] (2) Fluorescent dye loading: The growth medium in the CHO-TRPM8 cell culture medium in (1) was removed and replaced with 50 μL of HEPES-buffered saline (HBPS) solution containing 5 μL Fluor-8 and 5 μL Trypan red (10 mmol / L) at 37°C for 10 min, followed by fluorescent staining for 20 min.

[0054] (3) FLIPR detection: Add L-menthol (or menthone, menthyl acetate, WS-3, WS-5, WS-23, or anhydrous ethanol control sample) at a test concentration of 1.0 μg / mL to the wells of the flat-bottom microtiter plate containing HBPS buffer in step (2), record the fluorescence change value by FLIPR at an excitation / emission wavelength of 490 / 550 nm, and analyze it using FLIPRScreen Works 3.1 software.

[0055] (4) Data analysis: The raw fluorescence signal recorded in (3) was subjected to background correction and min-max normalization, and then ΔF / F was extracted. 0 As a quantitative indicator of the intensity of the cooling sensation, the percentage of TRPM8 channel activation is established. The EC 50 By comparing the EC of different cooling agents 50 The values ​​were used to conduct relative quantitative analysis of the cooling intensity.

[0056] The EC values ​​of this method for L-menthol, menthone, menthyl acetate, WS-3, WS-5, and WS-23 were calculated by changing the fluorescence intensity of different cooling agent solutions. 50 The values ​​were 8.6μg / mL, 9.2μg / mL, 8.0μg / mL, 8.7μg / mL, 7.9μg / mL, and 7.5μg / mL, respectively; the detection limits were 0.07μg / mL, 0.11μg / mL, 0.16μg / mL, 0.26μg / mL, 0.17μg / mL, and 0.08μg / mL, respectively.

[0057] Example 3

[0058] (1) Cell culture: CHO cells expressing the TRPM8 receptor clone were cultured at a concentration of 3.0 × 10 4Cells / well were seeded in 384-well flat-bottom microtiter plates. CHO cells were cultured at 37°C and 5% CO 2 and 95% relative humidity overnight.

[0059] (2) Fluorescent dye loading: The growth medium in the CHO-TRPM8 cell culture medium in (1) was removed and replaced with 50 μL of HEPES-buffered saline (HBPS) solution containing 5 μL Fluor-8 and 5 μL Trypan red (10 mmol / L) at 37°C for 10 min, followed by fluorescent staining for 20 min.

[0060] (3) FLIPR detection: Add L-menthol (or menthone, menthyl acetate, WS-3, WS-5, WS-23, or anhydrous ethanol control sample) at a test concentration of 10.0 μg / mL to the wells of the flat-bottom microtiter plate containing HBPS buffer in step (2), record the fluorescence change value by FLIPR at an excitation / emission wavelength of 480 / 540 nm, and analyze it using FLIPRScreen Works 3.1 software.

[0061] (4) Data analysis: The raw fluorescence signal recorded in (3) was subjected to background correction and min-max normalization, and then ΔF / F was extracted. 0 As a quantitative indicator of the intensity of the cooling sensation, the percentage of TRPM8 channel activation is established. The EC 50 By comparing the EC of different cooling agents 50 The values ​​were used to conduct relative quantitative analysis of the cooling intensity.

[0062] The EC values ​​of this method for L-menthol, menthone, menthyl acetate, WS-3, WS-5, and WS-23 were calculated by changing the fluorescence intensity of different cooling agent solutions. 50 The values ​​are EC 50 The values ​​were 8.7μg / mL, 9.1μg / mL, 8.1μg / mL, 8.4μg / mL, 7.7μg / mL, and 7.3μg / mL, respectively; the detection limits were 0.06μg / mL, 0.09μg / mL, 0.14μg / mL, 0.23μg / mL, 0.14μg / mL, and 0.08μg / mL, respectively.

[0063] Example 4

[0064] (1) Cell culture: CHO cells expressing the TRPM8 receptor clone were cultured at a concentration of 2.0 × 10 4 Cells / well were seeded in 384-well flat-bottom microtiter plates. CHO cells were cultured at 37°C and 5% CO2 and 95% relative humidity overnight.

[0065] (2) Fluorescent dye loading: The growth medium in the CHO-TRPM8 cell culture medium in (1) was removed and replaced with 40 μL of HEPES-buffered saline (HBPS) solution containing 5 μL Fluor-8 and 5 μL trypan red (10 mmol / L) at 37°C for 10 min, followed by fluorescent staining for 30 min.

[0066] (3) FLIPR detection: Add L-menthol (or menthone, menthyl acetate, WS-3, WS-5, WS-23, or anhydrous ethanol control) at a test concentration of 5.0 μg / mL to each well of the flat-bottom microtiter plate containing HBPS buffer in step (2), record the fluorescence change value by FLIPR at an excitation / emission wavelength of 480 / 540 nm, and analyze it using FLIPR Screen Works 3.1 software.

[0067] (4) Data analysis: The raw fluorescence signal recorded in (3) was subjected to background correction and min-max normalization, and then ΔF / F was extracted. 0 As a quantitative indicator of the intensity of the cooling sensation, the percentage of TRPM8 channel activation is established. The EC 50 By comparing the EC of different cooling agents 50 The values ​​were used to conduct relative quantitative analysis of the cooling intensity.

[0068] The EC values ​​of this method for L-menthol, menthone, menthyl acetate, WS-3, WS-5, and WS-23 were calculated by changing the fluorescence intensity of different cooling agent solutions. 50 The values ​​were 8.6μg / mL, 8.9μg / mL, 7.5μg / mL, 8.1μg / mL, 7.6μg / mL, and 7.4μg / mL, respectively; the detection limits were 0.03μg / mL, 0.05μg / mL, 0.08μg / mL, 0.14μg / mL, 0.05μg / mL, and 0.04μg / mL, respectively.

[0069] Test Example 1

[0070] In this test case, the State Key Laboratory of Food Resources and Exploration of Jiangnan University recruited 8 volunteers as the tasting panel, and the test standard was referred to the Q Grader standard for taste testing. The taste test protocol included each volunteer rinsing their mouth with 10 mL of 1% (fructose, glucose or sucrose) aqueous solution containing different concentrations of cooling agents for 10 seconds, then spitting it out, recording their sensory results on the sensory evaluation form, and taking a full rest between each cooling agent test.

[0071] The intensity of the cooling sensation of the cooling agent was scored on a scale of 0 to 9, with 0 being the least intense sensation and 9 being the strongest sensation at the beginning (the immediate sensation after spitting out the cooling agent after rinsing the mouth for 10 seconds) and 2 minutes after spitting out the cooling agent.

[0072] Before evaluating the test coolants and calibration panel, each panelist rinsed his mouth with 10 mL of a solution containing 10 μg / mL of L-menthol and 1% (fructose, glucose or sucrose) for 10 seconds and spit it out, setting the cooling intensity of the solution to 5. All subsequent sensory analyses of coolants were rated as values ​​equivalent to the 10 μg / mL L-menthol calibration value. Volunteers rested for 10 minutes after each evaluation. During the rest period, volunteers consumed unsalted crackers and water to clean up residual coolants. All volunteers were randomized to evaluate different samples to avoid any order effects.

[0073] Finally Figure 2 As shown, it was found that the order of cooling intensity evaluation by artificial sensory evaluation and the cell membrane method using TRPM8 channel was consistent, which was: WS-5 (5-point control value) > WS-3 > L-menthol > menthone > WS-23 > menthyl acetate. The results of the two methods were highly consistent, verifying the reliability and effectiveness of the TRPM8 channel cell membrane method in the evaluation of cooling intensity.

[0074] It should be understood that after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for detecting the cooling intensity of a cooling agent monomer based on a cell membrane method, characterized in that: The method comprises the following steps: Step (1), cell culture: Chinese hamster ovary cells stably expressing TRPM8 receptor were seeded into each well of a flat-bottom microtiter plate and proliferated to a predetermined confluency under culture conditions; Step (2), fluorescent dye loading: removing the cell culture medium in the flat-bottom microtiter plate of step (1), and adding a certain volume of HEPES-buffered saline (HBPS) buffer containing calcium ion fluorescent dye and trypan red for fluorescent staining; Step (3), FLIPR detection: Add a cooling agent and a control sample of a certain test concentration into the wells of the flat-bottom microtiter plate in step (2), and use a FLIPR to continuously record the change in fluorescence signal intensity; Step (4), data analysis: The fluorescence signal recorded in step (3) is background corrected and normalized, and the maximum fluorescence intensity change value ΔF / F0 is calculated as a quantitative indicator of the cooling intensity, that is, the activation percentage of the TRPM8 channel. A cooling agent concentration-maximum fluorescence intensity change value dose ΔF / F0-response curve is established to calculate the half effective concentration EC of the cooling agent. 50 , based on the half effective concentration EC of cooling agents 50 Conduct relative quantitative analysis of cooling intensity, and use the half effective concentration EC 50 The lower it is, the stronger the cooling sensation is.

2. The method for detecting the cooling intensity of cooling agent monomers based on the cell membrane method according to claim 1, characterized in that: The inoculation concentration of Chinese hamster ovary cells in step (1) is 2.0×10 4 ~4.0×10 4 cells / well, the culture medium was DMEM, and the culture conditions were 37°C, 5% CO2 and 95% relative humidity for 18-24h until the cell confluence was above 90%.

3. The method for detecting the cooling intensity of cooling agent monomers based on the cell membrane method according to claim 1, characterized in that: The volume of HBPS buffer added for fluorescent staining in step (2) is 30 to 50 μL, and the staining loading time ranges from 10 to 40 minutes.

4. The method for detecting the cooling intensity of cooling agent monomers based on the cell membrane method according to claim 1, characterized in that: In the step (2), the mass percentage of the calcium ion fluorescent dye 30mmol / L, trypan red 10mmol / L, and HEPES buffered saline in the HBPS buffer is 0.5%; The HEPES buffered saline contains: 10 g / L HEPES; 16 g / L NaCl; 0.74 g / L KCl; 0.27 g / L Na2HPO4.2H2O; and 2.0 g / L dextrose.

5. The method for detecting the cooling intensity of cooling agent monomers based on the cell membrane method according to claim 1, characterized in that: In step (3), the cooling agent includes L-menthol, menthone, menthyl acetate, WS-3, WS-5, WS-23, and the diluting solvent of the cooling agent is anhydrous ethanol; the control sample is anhydrous ethanol; The amount of the cooling agent added to the flat-bottom microtiter plate is 4-10 μL / well, and the dilution concentration of the cooling agent is set to 0.1-100 μg / mL.

6. The method for detecting the cooling intensity of cooling agent monomers based on the cell membrane method according to claim 1, characterized in that: The detection parameters of the FLIPR are: excitation wavelength 470-495 nm, emission wavelength 515-575 nm.

7. The method for detecting the cooling intensity of cooling agent monomers based on the cell membrane method according to claim 1, characterized in that: The normalization method in step (4) is a min-max normalization method; The formula for calculating the dose ΔF / F0 of the maximum fluorescence intensity change value is: ΔF / F0=(F-F0) / F0, where F0 is the initial fluorescence intensity and F is the fluorescence intensity after treatment The cooling agent concentration-maximum fluorescence intensity change value dose ΔF / F0-response curve formula is: ΔF / F0==K·C, where K is a proportional constant, the value range is K>0, and C is the concentration of the cooling agent.

8. Application of the method according to any one of claims 1 to 7 in the development of cool functional tobacco products, characterized in that: By EC 50 The value can be used to calibrate the amount of cooling agent added to achieve precise control of the product's cooling properties.

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